In 1952, Brànemark found out the Osseointegration phenomenon, in which titanium and bone were bound without mediating binding tissues, and with this as a turning point, implant made of pure titanium was clinically applied for the first time in 1965. A lot of implant treatment using Osseointegration phenomenon have been applied so far. However, a very long period of 3 to 6 months is required for titanium and bone to be bound. Heretofore, for the purpose of shortening this period by enhancing bone-affinity, many approaches such as redesigning hard matters being mainly mechanical material modification have been made, including modification to final surface treatment, vacuum evaporation of calcium or hydroxyapatite to titanium surface, or consideration of titanium alloy as a material, while no significant effect has been obtained so far. For example, in case of ceramics such as hydroxyapatite, there are problems such as reduction of coated layer or weakness to load due to physical characteristics. As for alloy, many cases have been considered so far, but in most of the cases, harmful tissue reactions have occurred, and therefore, only titanium and Ti6Al4V, being alloy thereof, is currently used.
Moreover, the relation between implant and surrounding mucosa is considered to be a scar tissue with small resistance to infection, which is different from that of tooth and gum. Therefore, in order to resolve this problem, approaches from hard matters have been considered as described in the above, including coating antibacterial agent to titanium surface. However, with such approaches from hard matters, improvement of anatomic/histological relationship between implant and surrounding mucosa are not considered.
Titanium is easily oxidized, and forms immediately dioxide in atmosphere and in water. As it is possible to degrade/detoxify almost all toxic substances by using photocatalytic activity of anatase crystals, which is one of titanium dioxide crystals, titanium is used for variously including degradation of bad smell such as sick house gas or acetaldehyde, or as anti-fungal agent. However, as the wavelength which anatase crystals can use is limited to the ultra-violet region, development of photocatalysts which can be used in a visible radiation region to awaited.
Furthermore, the following is proposed: a new implant containing osteopontin that can increase osseointegration rate and bone adhesion ratio, comprising an implant containing osteopontin in a formed state, wherein a material appropriate for use to a subject body in vivo is combined with an osteopontin in a releasable form (Published Japanese Translation of PCT International Publication No: 2002-500898); a technology controlling at a molecule level a construction of a metal compound to be bound, wherein in order to bind metal compound, and to provide a new protein, a protein fragment, a peptide or a derivative of a mutant thereof that is possible to bind metal compounds and to control the orientation or the sequence, a functional group having nitrilotriacetate construction is inserted to a characteristic stereo structure that the protein, the protein fragment, the peptide or the mutant thereof have (Japanese Laid-Open Patent Application No: 10-338700); or a method for preparing nanocrystals of semiconductors having specific crystal properties such as phase or configuration, by using self-organizing biological molecule modified so to have an amino acid oligomer that binds specifically to semiconductors (US Patent Application No. 2003/0073104 specification).
As mentioned above, when intending to confer higher capacity of titanium materials in the field of implant, not by modifying materials by hard matter, but with polymer such as protein being a soft matter that can bind flexibly to titanium surface, there is a problem that amino acid motif that recognizes/binds specifically to titanium surface does not exist in the nature. The object of the present invention is to provide a peptide sequence, a phage, an artifioial protein or a chimeric molecule with a binding ability to titanium necessary to confer higher capacity to titanium material with the use of soft matters, or a complex of a peptide, a phage, an artificial protein or a chimeric molecule having the peptide sequence and a functional peptide sequence, with titanium. Specifically, the present invention provides a functional titanium implant material wherein said peptide and a peptide or an artificial protein/a chimeric protein promoting calcification or bone growth/differentiation are bound to its surface, by which the osseointegration completes in a short period; or a functional titanium implant material wherein said peptide and a peptide or an artificial protein/a chimeric protein having high affinity to gum are bound to its surface, having a high resistance to bacterial infection, etc. Moreover, it provides a complex of the peptide or the artificial protein/the chimeric protein with oxidized titanium, or a complex of the complex with a low molecule compound such as chromophore, having a photocatalystic ability even in a visible radiation region, and a dioxide titanium pigment, wherein a chimeric protein/an artificial protein comprising the peptide and collagen gentle to skin, etc., are bound to its surface.
The present inventors made a keen study to solve the above problems. They brought into contact a population of phage wherein said phage of said population collectively express a library of different peptide sequences to titanium metal in an aqueous solution, recovered titanium bound to phage particles via peptide sequence by centrifugation, proliferated the obtained phage particles bound to titanium in E. coli, and repeated a panning operation comprising the contact of titanium with the proliferated titanium binding phage expressing a peptide sequence on phage particles and concentrating phage clones binding to titanium and obtained a phage library that might express a titanium binding peptide that recognizes specifically titanium.
The obtained phage library was cloned to examine the amino acid sequence expressed. By repeating the panning operation, it can be estimated that clones expressing sequences that can strongly bind to especially titanium had a major population in the phage library. The present inventors found that among 43 phage clones there were 33 clones expressing RKLPDAPGMHTW (peptide comprising an amino acid sequence shown in SEQ ID NO; 3), and that the binding ability to titanium of the phage clones expressing a peptide comprising an amino acid sequence shown in SEQ ID NO: 3 was much higher than that of the phage clone expressing a peptide comprising amino acid sequences shown in SEQ ID NOs: 16 to 38.
It is necessary to investigate whether the obtained clones expressing a peptide comprising an amino acid sequence shown in SEQ ID No: 3 are bound to titanium via the sequence they are expressing. It is possible to confirm whether the binding of the phage clone expressing a peptide comprising an amino acid sequence shown in SEQ ID NO:3 to titanium is bound specifically via the expressing sequence, with the use of QCM-D300 (Q-sense AB, Goeteborg), a device for quantification of interaction with a quartz crystal microbalance that can measure dissipation simultaneously. When a molecule elongated lengthwise like a phage, is bound perpendicularly to a crystal microbalance sensor, the viscoelasticity measured by dissipation increases significantly, while the frequency showing the binding level decreases. Further, even it is a very long elongated molecule, no significant increase of viscoelasticity is observed in contrast to the decrease of frequency, when bound horizontally to the side on which a crystal microbalance sensor was mounted. Actually, a result showing that a phage clone presenting a peptide comprising an amino acid sequence shown in SEQ ID NO: 3 was bound perpendicularly to titanium surface was obtained. It is probably the first example in the world, to have examined the binding form of a phage, by using a device for quantification of interaction with a quartz crystal microbalance, and it showed that this method was very useful for the analysis of a phage bound to the solid surface.
The titanium surface is oxidized immediately in water, and a hydroxy group binds to titanium atoms. It is estimated that the bound hydroxy group separates into a hydroxy group that bridges between two titanium atoms and a terminal hydroxy group bound with one titanium atom. As the polarity of the bridging hydroxy group and that of the terminal hydroxy group are different, they have a different pK. It is estimated that the bridging hydroxy group acts as an acid, and that the terminal hydroxy group acts as a base. It would be possible to control the binding of titanium and peptide by investigating how a peptide comprising an amino acid sequence shown in SEQ ID NO: 3 is bound specifically to titanium surface.
Generally, investigation of specificity of a peptide motif is conducted by identification of a residue playing an important role to the function due to the introduction of point mutation, or by refinement of functional region by an analysis of deletion mutant. In the former one, functional analysis of a series of alanine-substituted point mutants called alanine scanning is often performed. Substitution to alanine wherein methyl group has only one small side chain, and that has no charge, is thought to impair the function of the side chain of the amino acid residue. Alanine scanning was performed for the phage clone expressing a peptide comprising an amino acid sequence shown in SEQ ID NO: 3. A series of point-mutated phage clones expressing a peptide comprising amino acid sequences shown in SEQ ID NOs: 4 to 14 was prepared and the binding ability to titanium of each clone was examined. As a result, the binding ability of the point mutant to the 4th proline was the most significantly impaired, among those examined this time. Proline plays a role to curve widely the main chain of peptide or protein, similar to glycine. From this result, it was strongly suggested that the curve of the main chain in the 4th proline plays an important role for binding the peptide comprising an amino acid sequence shown in SEQ ID NO: 3 to titanium. Furthermore, as the binding ability of the point mutant to the 1st arginine and 5th aspartic acid among the amino acids being charged at the side chain was significantly impaired, it was suggested that these residues are mutually acted with the positive and negative charge of the titanium surface.
As a result of alanine scanning, a result supporting that the former part of SEQ ID NO: 2, region of SEQ ID NO: 1, plays an important role in the binding to titanium, was obtained. Therefore, a deletion mutant wherein 7th to 12th peptides comprising an amino acid sequence shown in SEQ ID NO: 3 are deleted, that is a phage clone expressing a peptide comprising an amino acid sequence shown in SEQ ID NO: 1 was prepared and the binding ability to titanium was investigated. As the binding ability to titanium was not affected by the deletion, it has been clarified that the amino acid sequence part shown in SEQ ID NO: 1 had a sufficient titanium binding ability.
When the peptide sequence shown in SEQ ID NO: 3 binds to titanium, the importance of the positive charge of the side chain of the 1st arginine is as described above, while there is still a possibility that it cooperates with an amino group at amino terminal of the main chain for binding to titanium. Therefore, an insert mutant (SEQ ID NO: 15) wherein alanine is inserted to amino terminal of the amino acid sequence shown in SEQ ID NO: 3 was prepared and the binding ability to titanium was investigated. As a result, increase of the binding ability to titanium was observed. As for the reason of the Increase of the binding ability, it can be considered that repulsion between the positive charge of the side chain of 2nd lysine of SEQ ID NO: 3 and the positive charge of an amino group at amino terminal of the main chain decreases due to the insertion of amino acid of one residue, and thus the structure of a peptide comprising an amino acid sequence shown in SEQ ID NO: 15 became more stable. Furthermore, this result shows that it is not always necessary that arginine is at the amino terminal when a peptide comprising an amino acid sequence shown in SEQ ID NO: 3 binds to titanium. This is an important knowledge showing that there is no limitation for primary construction as for the placement of SEQ ID NO: 1 or 3, when preparing a chimeric protein, an artificial protein or a synthetic peptide binding to titanium.
It is known that a number of hydroxy groups bind to titanium surface by hydrogen peroxide treatment. As it is mentioned above, it is thought that the interaction between a peptide comprising an amino acid sequence shown in SEQ ID NO: 1, 3 and titanium surface is dominated by the electrostatic interaction between the side chains of 1st arginine and 5th aspartic acid of SEQ ID NO: 1, 3 and the charge of the hydroxyl group bound to titanium. There is a possibility that the binding level of titanium and peptide can be controlled, if it would be possible to control the amount of hydroxyl group binding to titanium. In fact, the binding ability of the phage clone expressing SEQ ID NO: 3, treated with hydrogen peroxide, to titanium increased. This shows that by further adding hydroxy group to titanium surface by hydrogen peroxide treatment, it is possible to increase the binding level of the phage clone expressing SEQ ID NO: 3, the peptide of SEQ ID NO: 3, and the artificial protein/chimeric protein including thereof. Furthermore, by removing hydroxy group from titanium surface, it is expected to be possible to decrease the binding level of the phage clone expressing a peptide comprising an amino acid sequence shown in SEQ ID NO: 3, the peptide of SEQ ID NO: 3 and the artificial protein/chimeric protein including thereof. As for the method for removing hydroxy group from titanium surface, sodium fluoride treatment can be exemplified. By combining these methods, it is expected to be possible to control the binding level of the phage clone expressing SEQ ID NO: 3, the peptide of SEQ ID NO: 3, or the artificial protein/chimeric protein including thereof, to titanium.
Moreover, by investigating the binding specificity of peptides with a binding ability to titanium to metal materials, it was found they bind selectively to silver, silicon, besides titanium, and that they do not bind to gold, platinum, copper, iron, tin, zinc, chrome, etc. By using this binding specificity of metal materials, it might be possible to develop a certain pattern on gold basis of the functional compounds via titanium binding peptide, by for example performing patterning on gold basis with titanium, and by adding functional compounds, for example titanium binding peptide conjugating semiconductor nano particles, or artificial protein/chimeric protein including thereof.
The present inventors have thus completed the present invention according to the above knowledge.